Her platelets subsequently deteriorated further such that she started to need regular transfusions. An extensive workup, including measurement of vitamin B 12 and folate levels; serum protein electrophoresis; thyroid-stimulating hormone testing; serology for human immunodeficiency virus; and testing for hepatitis, Cytomegalovirus , Epstein-Barr virus, and parvovirus B19, was nondiagnostic.
A Coombs test was also negative, and abdominal ultrasound did not reveal any splenomegaly. Coagulation tests were normal. A bone marrow biopsy was performed, which showed erythroid hyperplasia with reduction of granulocytic precursors and marked reduction of megakaryocytes. There were no definitive morphologic features of myelodysplasia, and cytogenetic studies were also normal. The patient was treated with supportive measures, including platelet and occasional red cell transfusions, but became progressively more symptomatic over the next 6 months with worsening fatigue and more pronounced symptoms of dyspnea on exertion as well as episodes of abdominal pain.
A subsequent workup failed to reveal any cardiac or pulmonary cause for her symptoms, and abdominal imaging was also unremarkable.
At this time, her case was carefully reviewed, and the possibility of paroxysmal nocturnal hemoglobinuria PNH was considered. A peripheral blood sample was sent for flow cytometry to assess for PNH. Given the severity of her condition, she was started on specific anti-complement therapy, which resulted in dramatic improvement of her symptoms over the next 4 weeks.
However, she continued to require platelet transfusions owing to her bone marrow failure. Since the presence of a PNH clone in the setting of bone marrow failure is a marker for a high likelihood of response to immunosuppressive therapy, she was given a trial of cyclosporine. This case exemplifies many of the key points discussed in the previous article. In particular, it highlights the need to maintain an elevated index of suspicion for the possibility of PNH in the high-risk categories of hemolysis, thrombosis, and bone marrow failure, particularly when other clinical signs and symptoms of the disease are present.
This patient had severe fatigue that was out of proportion to the degree of anemia, and she also had episodes of abdominal pain, both of which are potential manifestations of PNH. Had this been considered during her initial workup, along with the fact that she was in one of the high-risk groups bone marrow failure , she could have been diagnosed and treated several months earlier, potentially avoiding many transfusions, not to mention the quality-of-life consequences.
Also, this patient was fortunate in that the diagnostic delay did not result in any of the potentially catastrophic thromboembolic complications of PNH. The dramatic improvement after complement-directed therapy rules out other explanations.
It highlights the fact that many of the symptoms of the disease may not be entirely due to hemolysis but may instead be caused by complement-mediated damage to other unprotected cell lines, such as granulocytes or platelets, which may be able to produce severe manifestations including cytokine-driven fatigue and platelet activation—driven thrombosis even with relatively small populations of cells. This theory, however, has not been well studied.
Vivek R. It is extremely severe, however, and we now have more effective therapies. So it has become particularly important for clinicians to diagnose it in a timely and effective manner. What, in your opinion, should hematopathologists know about this disease?
Sameer S. Talwalkar, MD ST Many hematopathologists are aware of the disease and how clinically important it is. The reason I believe it should be given more emphasis now is because there is an effective treatment. The onus is on the laboratories that are performing the PNH assay to provide quality results in a timely fashion. PNH is an ultra-rare disease, but it is life-threatening. Patients must receive an accurate and prompt diagnosis to avoid compromising treatment and outcome.
VS That is a very significant point. The role of hematopathologists is crucial because the final diagnosis is made based on the tests they run. How can a hematopathologist ensure that the tests are administered properly and the results are reported in a clear and appropriate format?
Are there any specific guidelines that hematopathologists should follow? ST In the past few years, the International Clinical Cytometry Society ICCS has published guidelines covering who should be tested, how testing should be performed, and how the results should be reported. As a hematopathologist, I routinely use these guidelines in day-to-day practice. A hematopathologist who is well trained in flow cytometry should be able to make the diagnosis of PNH.
Even laboratories with a basic 4-color flow cytometer can set up a PNH assay to provide accurate and timely results to physicians. Having said that, there are certain points to stress in terms of the quality and validation of this assay. I recommend running several negative samples and comparing these to a few known positives to see differences in staining and scatter properties of normal cells compared with PNH cells. From a clinical perspective, it is also very important to examine at least 2 different cell populations, ideally red cells and granulocytes.
ST First of all, the red cells in a patient who has untreated PNH undergo hemolysis, and therefore one may not get an accurate red cell clone size or may even get a false-negative result if looking at red cells alone. The second issue concerning red blood cells is that laboratories that do not use the CDa Glycophorin A antibody for measurement might obtain false-positive results as a result of not gating on a pure red cell population. The addition of granulocytes therefore increases not only sensitivity but also specificity of this assay by reducing the false positives.
VS I have worked with some laboratories that have sent reports based on testing of only the red cells. From what you are saying, that approach is suboptimal and contradicted by ICCS guidelines.
VS In terms of the sample, is it possible for clinicians to send bone marrow to test for PNH, or is peripheral blood preferred? In my experience, bone marrow samples can be used in the research setting, but not in the clinical laboratory.
Often these specimens have immature myeloid cells, which lack uniform expression of glycosylphosphatidylinositol GPI -anchored molecules or a high level of expression of these molecules.
The analysis will be flawed if it is based on immature cells that do not express GPI molecules uniformly. Another major disadvantage of using bone marrow samples involves patients with myelodysplastic syndrome, who have dysplasia in their myeloid lineage.
In these patients, it may not be possible to obtain an accurate analysis with a bone marrow specimen. There is a good reason for everything in those guidelines. There have been occasions when physicians have asked me questions about how to interpret the test report.
They can discuss red cells, granulocytes, and monocytes. Physicians who are less familiar with the details of PNH pathology and flow cytometry may sometimes wonder what the report is exactly saying. What are your thoughts on this issue? How can hematopathologists ensure they are providing a report that is clear, complete, and easy to understand?
ST It is very important for laboratories to communicate well in the report. Laboratories that are just starting to validate this assay or want to improve their approach can refer to the ICCS guidelines. The guidelines provide recommendations regarding how the results should be reported. My suggestion is that the results should be as concise and clear as possible. Rather, our reports, similar to those of laboratories that follow ICCS guidelines, provide an interpretation as to whether a PNH clone is present or absent.
With granulocytes, the clinical significance of the type II cells is uncertain. When the laboratory evaluates follow-up samples, the report should mention the previous clone size and highlight any changes in size that may be seen. This information might alert the clinician to the need for a change in treatment strategy. Lastly, reporting of small clone s is a bit challenging, especially without a good clinical history, since it can be difficult to interpret what a small clone really means clinically.
When small clones are found, it is important to document them in the pathology report and make recommendations in terms of patient follow-up. VS Those are all great points. As you said, the key is for the laboratory to break down the technical jargon so that the results are easily interpretable by clinicians who may not be familiar with the nomenclature associated with flow cytometry.
Reports that include interpretation can be very helpful and allow clinicians to more easily apply the results in the clinical context. It is also important, as you mentioned, for the report to include comments about small clones because the relevance of a small clone can be determined only over time. ST High-sensitivity analysis can detect small clones, up to 0. For laboratories that perform high-sensitivity PNH testing, it is very important to avoid false-positive results by repeating tests that are positive or having another technician perform the test, for reproducibility.
ST That is correct. One acquires more events so that it is possible to identify smaller clone sizes, typically as low as 0. VS It seems likely that most laboratories could set up a protocol for high-sensitivity analysis. It is a matter of taking a little extra time to count more cells. ST Correct. It is also very important to properly stain the cells and calibrate the instrument s. VS As we have discussed, the involvement of the hematopathologist is critical to the appropriate detection, diagnosis, and classification of PNH patients.
I have one final question for you, Dr Talwalkar. ST It is important for the hematopathologist to understand how critical it is to provide accurate and timely test results so that the patient can be started on treatment right away. To answer your question, it should be a team effort. Communication is critical. A good hematopathologist should be able to guide the clinical team in this effort.
Once the diagnosis is made, it should be communicated clearly and expeditiously to the clinician. Since PNH is a disease that can have fluctuations, it is important to have continued monitoring and communication between the clinicians and the hematopathologist. Dr Talwalkar has no real or apparent conflicts of interest to report.
Sharma, MD Assistant Professor of Medicine University of Louisville School of Medicine Louisville, Kentucky Abstract: Paroxysmal nocturnal hemoglobinuria PNH is an acquired clonal hematopoietic stem cell disorder with far-reaching multisystem effects that can lead to life-threatening consequences.
The Ham test checks whether red blood cells become more fragile when they are placed in mild acid. Flow cytometry suspends microscopic particles in a stream of fluid. The particles are then passed by an electronic detection apparatus. This tool counts and examines them. It can measure certain proteins associated with PNH to diagnose the disease. We treat some people who have PNH with blood transfusions, folic acid, and if appropriate, iron supplements. However, laboratory evidence of anemia and hemolysis may persist, regardless of the success of treatment.
Iron stores and serum erythropoietin concentrations should be examined in patients being treated with eculizumab who have persistent anemia to determine whether additional treatment to facilitate erythropoiesis is warranted. Parker CJ. Update on the diagnosis and management of paroxysmal nocturnal hemoglobinuria. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry.
Cytometry B Clin Cytom. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Pathophysiology, diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review. Eur J Haematol. Congenital CD59 deficiency. Hematol Oncol Clin North Am. Perkins S. Paroxysmal nocturnal hemoglobinuria. In: Kjeldsberg C. Practical Diagnosis of Hematologic Disorders, 5th ed. Screening and diagnostic clinical algorithm for paroxysmal nocturnal hemoglobinuria: expert consensus.
Trends in bone marrow sampling and core biopsy specimen adequacy in the United States and Canada: multicenter study.
Am J Clin Pathol. A neonate with Coombs-negative hemolytic jaundice with spherocytes but normal erythrocyte indices: a rare case of autosomal-recessive hereditary spherocytosis due to alpha-spectrin deficiency.
J Perinatol. Subscribe for Updates. Editorial Policy. Learn more. Cite this page. Topic Name. Message If ARUP Consult does not answer your test selection and interpretation questions, or if you would like to suggest ways to improve content or usability, please send a message to the Consult editorial staff.
ARUP will only use your email address to respond to your feedback. See the ARUP privacy policy for more information regarding email use. Leave this field blank. Quick Answers for Clinicians Which is the first-line test in patients with suspected paroxysmal nocturnal hemoglobinuria? What is the pathophysiology of paroxysmal nocturnal hemoglobinuria?
0コメント